Rhabdomyolysis is a significant cause of acute kidney injury (AKI), and apoptosis and necrosis are known to play major roles in rhabdomyolysis-induced AKI, but there is a fundamental knowledge gap of the factors that lead to their induction in the kidney. Continued existence of this gap represents a significant problem as AKI has a high mortality rate and there are very few therapeutic interventions to alter the clinical course of this disease. The long-term goal is to uncover the mechanisms involved in rhabdomyolysis-induced AKI for the development of novel therapeutics to protect the kidney. Ceramides regulate apoptosis and necrosis and are elevated in the kidney during AKI. The factors that regulate production of ceramides during kidney apoptosis and necrosis and whether ceramides lead to apoptotic versus necrotic kidney cell death are completely unknown. Likewise, there are many different ceramide species and the roles for particular ceramide species in AKI have not been determined. This proposal will answer these questions to achieve the objective of developing ceramides as novel therapeutic approaches for the treatment of AKI following rhabdomyolysis. Preliminary data demonstrate that: (i) C16-ceramide is generated via de novo synthesis during kidney cell apoptosis and blocking its generation inhibits apoptosis; (ii) the pro-apoptotic BCL-2 protein BAK is a key regulator of ceramide synthases (CerS) and long-chain ceramide generation during kidney cell apoptosis; (iii) acid sphingomyelinase (SMase) generated C26-ceramide occurs in kidney cell necrosis; and (iv) rat kidney cortical CerS and acid SMase are activated and specific ceramides elevated more than 5-fold in a rat model of rhabdomyolysis-induced AKI. This expanding and developing body of work has led us to propose the following hypothesis: nephrotoxic stimuli elevate specific species of kidney ceramides through CerS and SMase- mediated pathways, inducing kidney cell death and ultimately kidney failure. This hypothesis will be tested with three specific aims: (1) determine the mechanism by which BAK regulates CerS activity and generation of specific long-chain ceramides during kidney cell apoptosis; (2) determine the contribution of SMase-generated ceramide to kidney cell necrosis; and (3) determine the in vivo contribution of specific ceramides to rhabdomyolysis-induced AKI and kidney failure in rats. The approach is innovative because it utilizes novel methodologies to identify the specific role of individual ceramide species in AKI, namely the quantification of the individual ceramide species in the kidney cortex and in vivo knockdown of the expression of particular CerS isoforms specifically within the kidney. The proposed research is significant as it advances our current knowledge of mechanisms of AKI by identifying factors that regulate generation of specific ceramide species during kidney apoptosis and necrosis. Ultimately such knowledge has the potential to greatly improve the treatment of AKI.